Reg-like protein

ABSTRACT

Methods of detecting the presence of a tumor or a cancerous condition includes detecting the expression of RELP proteins, related polypeptides and proteins, or nucleic acid molecules indicative of such expression. These methods include antibody-based assays and molecular diagnostic assays such as PCR-based methods.

BACKGROUND

[0001] The invention relates to proteins whose presence, absence,concentration in biological samples, or expression level can be used toindicate the presence or absence of tumors, cancer, and relatedconditions as well as related prognoses and diagnoses.

[0002] Tumors are abnormal masses of tissue. When tumors proliferateuncontrollably, they are said to become malignant. This condition isgenerally referred to as a cancer. Numerous methods are used todetermine when a patient has developed a tumor and when the conditionhas become cancerous. The identification or quantitation of varioustumor or cancer markers is one desirable means for making suchdeterminations.

[0003] Broadly, a “marker” is any property that can be used todistinguish cancer from normal tissues and from other disease states.The markers' presence is then a basis for classification. Morespecifically, the term is used to denote particular molecules that areamenable to assay. Serum markers, as the name implies, are markers thatare readily assayed in the serum of a patient. Typically, they aresecreted proteins or cell receptors that are abundant in tumor cellswell beyond their presence (or total absence) in normal cells andtissues. Examples include PSA, CEA, and AFP.

[0004] A more expansive consideration of tumor and cancer markersincludes the detection of tumors and cancer from the nucleic acidsproduced in various cells (as well as other materials that are relatedto nucleic acids). Cancer is generally considered to be a disease ofmultiple mutations. Thus, detection of the mutations at the molecularlevel offers the prospect of more direct and more reliable diagnosesthan was possible with some of the older cancer markers. Thus, it isappropriate to consider a nucleic acid sequence that is indicative ofthe mutation that causes or occurs with the cancerous condition to be acancer marker. The ability to conduct nucleic acid analyses does notvitiate the value of serum markers, however. Each may have anappropriate role to play in the diagnosis, staging, and treatmentmonitoring of a patient.

[0005] Discovering genes that encode cancer-associated antigens andevents also opens the door to genetic intervention against cancer cellproliferation. The accurate and consistent use of a cancer marker todifferentiate cancerous from normal tissue, not only has diagnosticpotential, but is also desirable for treatment and prognosis. Therefore,such markers continue to be sought.

[0006] The reg proteins, which belong to the C-type lectin superfamily,are secreted proteins of about 20 kD in size. They are found in normaland malignant tissues of the gastrointestinal tract, in the pituitaryand in regenerating neurons. Reg expression associates with cellproliferation, migration and differentiation (Chiba T et al., 2000, JGastroenterol 35 Suppl 12:52, Levine J L, 2000, Surg Res 89:60,Otonkoski T et al., 1994, Diabtets 43:1164, Bemard-Perronese FR, 1999, JHistochem Cytochem 47:863). The known reg genes cluster on humanchromosome 2p12. The first characterized member of the reg proteinfamily was Reg 1α, which was isolated from rat regenerating pancreaticislets (Terazono et al., 1988). Subsequently, cDNAs encoding for fouradditional human reg proteins, and the corresponding mouse and ratorthologs, have been cloned (Watanabe et al., 1990; Lasserre et al.,1992; Bartoli et al., 1993; Rafaeloff et al., 1997). They exertmitogenic activity to subsets of epithelial and neuroectodermal cells(Katsumata et al., 1995, Zenilman et al., 1996; 1997; 1998; Livesey etal., 1997). A growth signal transducing receptor for rat reg1 proteinswas recently described. The receptor is encoded by a gene homologous tohuman multiple exostoses gene. It was found to have been expressed, inaddition to pancreatic islets, in various tissues including kidney,liver, gut, the adrenal and pituitary glands (Kobayashi S et al. 2000).

[0007] Identification, isolation, and use of new tumor and cancermarkers remain important in the diagnosis, treatment and prevention ofcancer.

BRIEF SUMMARY OF THE INVENTION

[0008] The invention is an isolated nucleic acid molecule that encodesRELP protein. The molecule can be a nucleic acid molecule of Seq ID No1, a nucleic acid molecule encoding a protein having at least a 70%identity to a polypeptide comprising amino acids of SEQ ID NO:2.

[0009] The invention also encompasses a nucleic acid molecule that iscomplementary to the molecule that encodes a protein having at least 70%identity to Seq. ID No. 2, a nucleic acid molecule of at least 15sequential bases of the nucleic acid sequence of Seq. ID No. 1, or anucleic acid molecule that hybridizes under stringent conditions to thenucleic acid sequence molecule of Seq. ID No. 1.

[0010] In another aspect of the invention, isolated RELP is presented.

[0011] In yet another aspect of the invention methods of detecting thepresence of a tumor or a cancerous condition includes detecting theexpression of polypeptides, proteins, or nucleic acid molecules havingthe sequences described above and correlating the presence orconcentration of such molecule in a biological sample with the presenceor absence of said tumor or cancerous event.

[0012] In yet another aspsect of the invention, antibodies that binds tothe RELP and functional equivalents thereof are presented.

[0013] In yet another aspect of the invention, kits for detecting thepolypeptides, proteins, or nucleic acid sequences described above arepresented.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is the nucleic acid sequence of the cDNA that encodes forRELP (Seq. ID No. 1).

[0015]FIG. 2 is the amino acid sequence of RELP (Seq. ID No. 2).

[0016]FIG. 3 is the nucleic acid sequence of the cDNA that encodes forRELP signal protein (Seq. ID No. 3).

[0017]FIG. 3a is the amino acid sequence of RELP signal protein (Seq. IDNo. 4).

[0018]FIG. 4 is a scaled schematic representation of the RELP gene.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Definitions:

[0020] The term “protein superfamily” as used herein refers to proteinswhose evolutionary relationship may not be entirely established or maybe distant by accepted phylogenetic standards, but show similar threedimensional structure or display unique consensus of critical aminoacids. The term “protein family” as used herein refers to proteins whoseevolutionary relationship has been established by accepted phylogenicstandards.

[0021] As used herein, the term nucleic acid sequence includes DNAs orRNAs as described above that contain one or more modified bases. Thus,DNAs or RNAs with backbones modified for stability or for other reasonsare “nucleic acid sequences” as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritylated bases, to name just two examples, are nucleicacid sequences as the term is used herein. It will be appreciated that agreat variety of modifications have been made to DNA and RNA that servemany useful purposes known to those of skill in the art. The termnucleic acid sequence as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of nucleic acid sequences,as well as the chemical forms of DNA and RNA characteristic of virusesand cells, including simple and complex cells, inter alia. Nucleic acidsequences embraces short nucleic acid sequences often referred to asoligonucleotide(s).

[0022] As used herein, a “functional derivative” of RELP is a compoundthat possesses a biological activity (either functional or structural)that is substantially similar to the biological activity of RELP. Theterm “functional derivatives” is intended to include the “fragments,”“variants,” “degenerate variants,” “analogs” and “homologues” or to“chemical derivatives” of RELP. A molecule is “substantially similar” toRELP if both molecules have substantially similar structures or if bothmolecules possess similar biological activity.

[0023] A newly identified protein, “RELP” (Reg Like Protein), ischaracterized in this specification. Nucleic acids (including, forexample, cDNA) encoding for this protein have been isolated and clonedand uses for this protein in cancer diagnostics are presented. The genestructure and its chromosomal location are presented, and the tissuedistribution of its expression is described. Additionally, antibodiesthat bind to this protein have been prepared and methods for their usehave been devised. The murine homologue of RELP was also cloned andcharacterized.

[0024] All nucleic acid sequences described in this specification areshown in the 5′→3′ direction unless otherwise indicated.

[0025]FIG. 1 shows the nucleic acid sequence of a cDNA (Seq. ID No. 1)used to produce RELP. The RELP cDNA encodes a 158-amino acid proteinwith a putative 22-amino acid signal peptide (FIG. 3). The molecularweight of RELP is about 18 kd, and the isoelectric point was calculatedas 9.128. The aminoterminus of RELP is highly hydrophobic and contains acleavable signal sequence of 22 aminoacids. Human Reg proteins are51-87% identical and 55-87% similar to each other, whereas RELP is32-37% identical and 42-47% similar to them.

[0026] The primary structure of RELP is similar to that of the subgroupof C-type lectin superfamily of proteins, which contain a singlecarbohydrate-recognition (CRD) domain. The CRD-associated four conservedand two optional cysteines involved in intramolecular disulphide bondsare all conserved in RELP. Residues 50-53 represent a putativeN-glycosylation site. The secondary structure of RELP is similar to thatof human Reg1α and the global folds of these proteins appear to berelated. The amino acid sequence of RELP is shown in FIG. 2 (Seq. ID No.2).

[0027] The RELP gene resides on chromosome 1 band p12-13.1 and spansabout 17,500 base pairs. It is comprised of seven exons. FIG. 4 shows aschematic representation of the gene with the distance between exonsscaled. The location of each exon is shown in Roman numerals.

[0028] Expression of RELP in normal tissues: RELP message is highlyexpressed in a subset of epithelial cells in the small intestine. Thissubset of cells represents the intestinal neuroendocrine cells (verifiedby colocalization of chromogranin). RELP mRNA is also seen in thestomach, various parts of the colon, where it is localized in theepithelial cells in the crypt bottom, the pancreas, the prostate and thetestis.

[0029] Expression of RELP in diseased tissues: RELP is ectopicallyabundantly expressed in mucinous tumors originating from various organs,such as ovary, stomach, colon, breast and pancreas. The expression ofRELP mRNA appears to be extremely high in mucinous ovarian tumors. On aprotein level a high, uniform expression is seen in the epithelial cellsfrom mucinous ovarian, stomach, colon and breast tumors. Intraductalmucinous pancreatic tumors also express RELP. These tumors are emergingas a newly identified entity of pancreatic disorders that predisposerecurrent pancreatitis. They are probably apt to become malignant.

[0030] Biological samples from a subject are used to determine whethercancer cells are present in the subject. Examples of suitable samplesinclude blood and biopsy material. One method of diagnosis is to exposeRNA from cells in the sample to a labeled probe that is capable ofhybridizing to the RELP gene transcript, or a fragment thereof, understringent conditions. Of course, the hybridizing conditions are alteredto achieve optimum sensitivity and specificity depending on the natureof the biological sample, type of cancer, method of probe preparation,and method of tissue preparation.

[0031] After contacting the sample with the probe, the next step isdetermining whether the probe has hybridized with nucleotide sequencesof the mRNA from the sample, from which the expression of the RELP geneis inferred, the presence at elevated levels being diagnostic of cancer.

[0032] Another diagnostic method is to contact a sample with antibodiesdirected to antigenic (i.e. RELP) peptides. These antibodies are usefulin the development of very specific assays for the detection of RELPantigen, and allow the tests to be carried out in many differentformats. Preferably, the antibodies are labeled monoclonal antibodies.Since RELP is a secreted molecule, detecting RELP antigen in bodyfluids, such as serum, plasma, cyst fluids, pancreatic juice, and urinecan be used to detect or follow-up RELP-expressing cancers. Typically,the protein is expressed between 100 and 1000 times in diseased tissues(as described above) compared with its normal expression levels.Accordingly, serum levels of 200 to 1000% those of normal levels will bedetected in the serum assays of this invention. Most typically, a serumlevel of about 250% that of normal RELP levels can be expected inpatients with colon cancer. Likewise, in molecular diagnostic tests inwhich mRNA expression levels are assayed, expression levels that are 150to 1000% those of normal levels indicate disease.

[0033] Purified biologically active RELP may have several differentphysical forms.

[0034] RELP may exist as a full-length nascent or unprocessedpolypeptide, or as partially processed polypeptides or combinations ofprocessed polypeptides. The full-length nascent RELP polypeptide may bepostranslationally modified by specific proteolytic cleavage events thatresults in the formation of fragments of the full length nascentpolypeptide. A fragment, or physical association of fragments may havethe full biological activity associated with RELP however, the degree ofRELP activity may vary between individual RELP fragments and physicallyassociated RELP polypeptide fragments.

[0035] Since there is a substantial amount of redundancy in the variouscodons that code for specific amino acids, this invention is alsodirected to those DNA sequences that contain alternative codons thatcode for the eventual translation of the identical amino acid. Forpurposes of this specification, a sequence bearing one or more replacedcodons will be defined as a degenerate variation. Also included withinthe scope of this invention are mutations either in the DNA sequence orthe translated protein, which do not substantially alter the ultimatephysical properties of the expressed protein. For example, substitutionof aliphatic amino acids alanine, valine, leucine and isoleucine;interchange of the hydroxyl residues serine and threonine, exchange ofthe acidic residues aspartic acid and glutamic acid, substitutionbetween the amide residues asparagine and glutamine, exchange of thebasic residues lysine and arginine and among the aromatic residuesphenylalanine, tyrosine may not cause a change in functionality of thepolypeptide. Such substitutions are well known and are described, forinstance in Molecular Biology of the Gene, 4^(th) Ed. Bengamin CummingsPub. Co. by Watson et al.

[0036] It is known that DNA sequences coding for a peptide may bealtered so as to code for a peptide having properties that are differentthan those of the naturally occurring peptide. Methods of altering theDNA sequences include, but are not limited to site directed mutagenesis,chimeric substitution, and gene fusions. Site-directed mutagenesis isused to change one or more DNA residues that may result in a silentmutation, a conservative mutation, or a nonconservative mutation.Chimeric genes are prepared by swapping domains of similar or differentgenes to replace similar domains in the RELP gene. Similarly, fusiongenes may be prepared that add domains to the RELP gene, such as anaffinity tag to facilitate identification and isolation of the gene.Fusion genes may be prepared to replace regions of the RELP gene, forexample to create a soluble version of the protein by removing atransmembrane domain or adding a targeting sequence to redirect thenormal transport of the protein, or adding new post-translationalmodification sequences to the RELP gene. Examples of altered propertiesinclude but are not limited to changes in the affinity of an enzyme fora substrate or a receptor for a ligand. All such changes of the nucleicacid sequence or polypeptide sequences are anticipated as usefulvariants of the present invention so long as they retain theirfunctionality consistent with the original use of the nucleic acidsequence or polypeptide sequence of the present invention as describedherein.

[0037] Identity or similarity, as known in the art, are relationshipsbetween two or more polypeptide sequences or two or more nucleic acidsequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolypeptide or nucleic acid sequence sequences, as the case may be, asdetermined by the match between strings of such sequences. Both identityand similarity can be readily calculated (Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991). While there exist a numberof methods to measure identity and similarity between two nucleic acidsequences or two polypeptide sequences, both terms are well known toskilled artisans (Sequence Analysis in Molecular Biology, von Heinje,G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H.,and Lipman, D., (1988) SIAM J. Applied Math., 48, 1073.

[0038] Methods commonly employed to determine identity or similaritybetween sequences include, but are not limited to those disclosed inCarillo, H., and Lipman, D., (1988) SIAM J. Applied Math., 48, 1073.Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in computer programs. Preferred computer programmethods to determine identity and similarity between two sequencesinclude, but are not limited to, GCG program package (Devereux, J., etal., (1984) Nucleic Acids Research 12(1), 387), BLASTP, BLASTN, andFASTA (Atschul, S. F. et al., (1990) J. Molec. Biol. 215, 403).

[0039] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Many amino acids, including the terminal amino acids, may be modified ina given polypeptide, either by natural processes, such as processing andother post-translational modifications, but also by chemicalmodification techniques which are well known to the art. Even the commonmodifications that occur naturally in polypeptides are too numerous tolist exhaustively here, but they are well described in basic texts andin more detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Amongthe known modifications which may be present in polypeptides of thepresent are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Such modificationsare well known to those of skill and have been described in great detailin the scientific literature. c.f. PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993).

[0040] Included within the scope of the invention are nucleic acidsequences that are at least 70% identical over their entire length to anucleic acid sequence encoding the polypeptide having the amino acidsequences set out herein, and nucleic acid sequences which arecomplementary to such nucleic acid sequences. Alternatively, highlypreferred are nucleic acid sequences that comprise a region that is atleast 80% identical, more highly preferred are nucleic acid sequences atcomprise a region that is at least 90% identical, and among thesepreferred nucleic acid sequences, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% identity are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the most preferred. The nucleic acid sequences whichhybridize to the hereinabove described nucleic acid sequences in apreferred embodiment encode polypeptides which retain substantially thesame biological function or activity as the polypeptide characterized bythe RELP amino acid sequences set forth herein. Preferred embodiments inthis respect, moreover, are nucleic acid sequences that encodepolypeptides that retain substantially the same biological function oractivity as the mature polypeptide encoded by the DNA of Seq. Id No. 1.The present invention further relates to nucleic acid sequences thathybridize to the herein above-described sequences. In this regard, thepresent invention especially relates to nucleic acid sequences thathybridize under stringent conditions to the herein above-describednucleic acid sequences. As herein used, the term “stringent conditions”means hybridization will occur only if there is at least 95% andpreferably at least 97% identity between the sequences.

[0041] Nucleic acid sequences of the invention may be used as ahybridization probe for RNA, cDNA and genomic DNA to isolate full-lengthcDNAs and genomic clones encoding the sequences of RELP set forth hereinand to isolate cDNA and genomic clones of other genes that have a highsequence similarity to them. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less. For example, the codingregion of the gene of the invention may be isolated by screening usingthe known DNA sequence to synthesize an oligonucleotide probe. A labeledoligonucleotide having a sequence complementary to that of a gene of thepresent invention is then used to screen a library of cDNA, genomic DNAor mRNA to determine to which members of the library the probehybridizes.

[0042] The polypeptides of the present invention include the polypeptideof Seq. ID No. 2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% identity to the polypeptide of Seq.ID No. 2, preferably at least 80% identity to the polypeptide of Seq. IDNo. 2, and more preferably at least 90% similarity (more preferably atleast 90% identity) to the polypeptide of Seq. ID No. 2 and still morepreferably at least 95% similarity (still more preferably at least 97%identity) to the polypeptide of Seq. ID No. 2 and also include portionsof such polypeptides with such portion of the polypeptide generallycontaining at least 30 amino acids and more preferably at least 50 aminoacids. Representative examples of polypeptide fragments of theinvention, include, for example, truncation polypeptides of Seq. ID No.2 or of variants or derivatives thereof, except for deletion of acontinuous series of residues (that is, a continuous region, part orportion) that includes the amino terminus, or a continuous series ofresidues that includes the carboxyl terminus or, as in double truncationmutants, deletion of two continuous series of residues, one includingthe amino terminus and one including the carboxyl terminus. Alsopreferred in this aspect of the invention are fragments characterized bystructural or functional attributes of the polypeptide characterized bythe sequences of Seq. ID No. 2.

EXAMPLES Example 1 Cloning and sequencing of cDNAs

[0043] An EST that was abundantly expressed in mucinous ovariantumor-derived libraries was identified. A full length cDNA insert,encoding for the predicted preprotein based on the EST was acquired,cloned into the pSport vector, and verified by sequencing. Blasting withthe human RELP cDNA in the NCBI EST data base yielded three highlyhomologous mouse sequences. The corresponding clones (IMAGE clone IDs717371, 1079498 and 1096767) were acquired and sequenced. The putativemouse orthologue for RELP was cloned into the XbaI site in thepGEMA1bSVPA vector. Murine RELP and the nucleic acid that codes for itwas found to have 66% amino acid and 70% nucleotide sequence identityrespectively to human RELP and its associated nucleic acid. Murine RELPwas also found to have 43-45% similarity and 32%-37% identity to othermouse Reg sequences. The nucleic acid sequence of cDNA used to expressit is shown in FIG. 1 (Seq. ID No. 1).

Example 2 Antibodies

[0044] A C-terminal RELP-derived peptide was synthesized(CAEMSSNNNFLTWSSNE Seq. ID No. 5), coupled to keyhole limpet hemocyanin,and used to immunize rabbits for production of polyclonal antibodies.The sera were tested for reactivity against the corresponding peptidewith ELISA, and the positive batches were affinity-purified. Thepurified antibody specifically detected the protein that has the peptideepitope in tissue sections. This was verified by complete abolishment ofthe signal if the corresponding peptide is added simultaneously with theantibody. In addition to this polyclonal antibody, which works well inimmunohistochemistry, monoclonal antibodies able to detect the proteinin its natural fold were produced. To produce monoclonal antibodies, apurified antigen, produced in mammalian cells to ensure natural fold andposttranslational modifications, was generated. The antigen, RELP-IgGconstant part fusion protein, was expressed in mouse myeloma cells, andthe secreted protein was purified using the Fc part as bait. Thispurified antigen was recognized in Western blot by the C-terminalpolyclonal antibody, and by five other anti-RELP peptide antibodies(Below, Seq. ID No.6-Seq. ID No.11). The antigen was used to generatemouse monoclonal antibodies against RELP by selecting out of thepositive clones those that produced antibodies that reacted against RELPinstead of the IgG constant part.

[0045] Kits for the clinical identification of RELP can be readilyfashioned employing these and similar antibodies. Such kits wouldinclude antibodies directed to RELP identification, appropriateindicator reagents (e.g., enzymes, labels, and the like), and(optionally) other reagents useful in the clinical application of such akit such as dilution buffers, stabilizers, and other materials typicallyused in such assays. The kits would be used to detect RELP in bodyfluids to screen or follow-up RELP expressing cancers, and to screen thepresence of RELP protein in tissue samples. CYGYFRKLRNWSDAELECQSYGNGASeq.ID No.6 WIDGAMYLYRSWSGKSMGGNKHC Seq.ID No.7 CAEMSSNNNFLTWSSNE Seq.IDNo.8 CAEMSSNMNFLTWSSNECNKRQHFLCKYR Seq.ID No.9CEYISGYQRSQPIWIGLHDPQKRQQWQ Seq.ID No.10 CQSYGNGAHLASILSLKEASTIA Seq.IDNo.11

Example 3 Double Immunofluorescence Staining

[0046] Tissue sections of normal duodenal mucosa were double stainedwith the polyclonal peptide antibody against RELP (1:30; 25 μg/ml) and amonoclonal antibody against chromogranin A (1:5000; 0.2 μg/ml Chemicon,Temecula, Calif.) followed by tetramethylrhodamineisothiocyanate-conjugated swine anti-rabbit immunoglobulins (DAKO) andfluorescein isothiocyanate (FITC)-conjugated goat anti-mouseImmunoglobulins (ICN/Cappel). For control stainings primary antibodieswere replaced with the IgG fractions of normal rabbit and mouse sera.

[0047] The colocalization of RELP and chromogranin A indicates that theRELP-expressing cells in the duodenum belong to the neuroendocrinepopulation.

Example 4 In situ Hybridization

[0048] Formalin fixed paraffin embedded tissue samples were cut into 5-7mμ thick sections, mounted on silane coated glass slides, and incubatedat 37° C. over night and at 65° C. for 30 min before deparaffinatingtwice for 10 min in xylene. Thereafter the samples were rehydratedthrough a graded series of ethanol solutions (100 to 70%), and rinsedtwice for 5 min in phosphate buffered saline (PBS pH 7.0), treated twicefor 5 min with 0.1 mol/L glysine in PBS, permeabilized for 15 min with0.3% Triton X-100 in PBS. The sections were treated with proteinase K(Finnzymes, Helsinki, Finland) treatment (μg/ml, in TE buffer; 100mmol/L Tris-HCl, 50 mmol/L EDTA, pH 8.0) at 37° C. for 30 min, postfixedin 3% paraformaldehyde in PBS at 4° C. for 5 min and rinsed twice inPBS. Positive charges were blocked by soaking the slides in 0.25% (v/v)acetic anhydride, 100 mmol/L triethanolamine, pH 8.0, twice for 5 min.The slides were equilibrated in 4×SSC, 50% (v/v) deionized formamide at37° C. for 10 min. Probes were prepared by ligating a PCR-amplified 0.4kb RELP cDNA insert into the pCR-II vector using a TA cloning kit(Invitrogen, San Diego Calif., USA). The templates for RELP antisense orsense RNA probes were generated by linearizing the appropriate vectorconstruct (in 3′ to 5′ direction or 5′ to 3′ direction, respectively).

[0049] An RNA Labeling Kit (Boehringer-Mannheim) was used to generatedigoxygenin labeled RNA probes by in vitro transcription. Thehybridization was performed overnight at 45° C. using a hybridizationmixture containing 1× Denhart's solution (0.2 g/L Ficoll Type 400,Pharmacia), 0.2 g/L polyvinylpyrrolidone, 0.2 g/L bovine serum albumin(fraction V; Sigma), 40% formamide, 10% dextran sulfate, 4×SSC, 10mmol/L dithiothreitol, 1 mg/mL yeast tRNA, 1 mg/mL herring sperm DNA and300 ng/mL digoxygenin-labeled RNA probe. After hybridization, the tissuesections were washed at 37° C. twice for 5 min in 2×SSC and once for 15min in 60% formamide, 0.2×SSC, followed by two 5 minute rinses in 2×SSCat room temperature and two 10 minute washes in 100 mmol/L Tris-HCl, pH8.0, 150 mmol/L NaCl. The signal detection was carried out using 1:250alkaline phosphatase-conjugated sheep antidigoxygenin fab fragments(Boehringer Mannheim). The signal was visualized by incubating thesections with NBT/BCIP Stock Solution (Boehringer Mannheim) for 1.5hours.

[0050] Small numbers of RELP-positive cells were seen in the gastricmucosa and in exocrine pancreas. In normal colon, RELP was localized inepithelial cells in the bottom of the crypts. A strong RELP mRNA signalwas seen in the cytoplasm of selected cells in the duodenal mucosa whilemost of the epithelium was negative. In mucinous cancers from ovary,stomach, colon and breast the RELP mRNA was also detected in theepithelial cells. The visualization of the RELP-specific mRNA confirmedthat the RELP protein was expressed by these cells.

Example 5 Immunohistochemistry

[0051] An affinity-purified polyclonal antibody against the C-terminalpeptide of RELP was used for the immunohistochemical detection andlocalization of RELP. Four μm sections from formalin-fixed and paraffinembedded normal and tumor tissue, obtained from the archives of theDepartment of Pathology, University of Helsinki, were mounted on3-aminopropyl-triethoxy-silane (APES, Sigma, St. Louis, Mo., U.S.A)coated slides. The sections were deparaffinized and rehydrated in gradedconcentrations of ethanol and treated with methanolic peroxide (0.5%hydrogen peroxide in absolute methanol) for 30 minutes at roomtemperature to block the endogenous peroxidase activity. Antigenretrieval was done in a microwave oven twice for 5 minutes (650W). AnElite ABC Kit (Vectastain, Vector Laboratories, Burlingame, Calif.,U.S.A) was used for immunoperoxidase staining. The RELP antibody wasused at an optimal dilution of 1:2000. Both the biotinylated secondantibody and the peroxidase-labeled avidin-biotin complex were incubatedon the sections for 30 minutes. The dilutions were made in PBS (pH 7.2),and all incubations were carried out in a moist chamber at roomtemperature. Between the different staining steps the slides were rinsedthree times with PBS. The peroxidase staining was visualised with a3-amino-9-ethylcarbazole (Sigma) solution (0.2 mg/ml in 0.05 M acetatebuffer containing 0.03% hydrogen peroxide, pH 5.0) at room temperaturefor 15 minutes. Finally, the sections were lightly counterstained withMayer's haematoxylin and mounted with aqueous mounting media (Aquamount,BDH). In control experiments the primary antibodies were replaced withthe IgG fraction of normal rabbit serum or the primary antibody waspreabsorbed with the RELP peptide. These stainings indicated thepresence of the RELP protein in a subset of duodenal epithelial cells,in a subset of stomach mucosal epithelial cells, in a subset of exocrinepancreatic ductal cells, in colon crypt bottom cells, in a subset ofmammary ductal epithelial cells, and in the epithelial cells of benignand malignant mucinous tumors originating from ovary, stomach, colon,breast, and pancreas, while the stroma remained completely negative. Theabundant and uniform expression of RELP protein in the epithelial cellsfrom mucinous tumors further supports the use of RELP as a tumor marker.As a secreted protein RELP can be measured from the serum or plasma.Moreover, anti-RELP antibodies might prove useful in detecting solitarytumor cells in tissue samples and cytologic specimen.

Example 6 Structure and Nucleotide Sequence of the Gene

[0052] RELP cDNA comprises 1517 nucleotides, and the protein codingregion is made up of 476 bp of nucleotides encoding a preprotein of 158amino acids. The 5′untranslated and 3′ untranslated regions contain 440and 601 nucleotides respectively. The first methionine (nt 441-443) ispreceded by a Kozaks' consensus translational start site. (Kozaksequence AAG before initiating methionine). A polyadenylation signal(AATAAA) is located 510 bp downstream of the termination codon. The genestructure of the protein was deduced by the analysis of genomicdatabases in the public domain. The missing base pairs flanking ends ofthe randomly ordered fragments of the genomic data base were acquired bysequencing these areas of the physical genomic RELP sequence.

[0053] A human genomic PAC clone containing the genomic RELP sequencewas obtained from GenomeSystemsInc (St. Louis, Mo.). NS3516 bacterialcells were transformed with the PAC plasmid containing a genomic insertof about 120 kb. Plasmid DNA was isolated using EndoFree Plasmid MaxiKit (Qiagen, Germany). The genomic sequence was amplified by PCR usingRELP-specific primers flanking the missing sequence data.

[0054] The primers used were as follows: CAGCTGTGCTCCTGGATGGT Seq.IDNo.12 TGGTCGGTACTTGCACAGGA Seq.ID No.13 CTCCTATTGCTGAGCTGCCT Seq.IDNo.14 ATTCGTTGCTGCTCCAAGTT Seq.ID No.15 TTCCAGAAGCATGCGGCTG Seq.ID No.16ACAGGAAGTGTTGGCGCTT Seq.ID No.17 ATGGCTTCCAGAAGCATGC Seq.ID No.18CTATGGTCGGTACTTGCACA Seq.ID No.19 CTTGCTCTATGGTCGGTACT Seq.ID No.20ACTGGGACCACTGGAGACACT Seq.ID No.21 GAGACACTGAAGAAGGCAG Seq.ID No.22AGACCCAGCTGTTTCATAGG Seq.ID No.23 AATGGAGAGAGGGCAGAAGG Seq.ID No.24TGATATCATCATGAGACCCAGCT Seq.ID No.25 AGACAGTCATCCATTTGCCCA Seq.ID No.26TGGGCAAATGGATGACTGTCT Seq.ID No.27 CTCTAGAATCCAACAAAACTC Seq.ID No.28TGCCAGACCAGGATCTGTACA Seq.ID No.29 ATCCATATCGGCTGGCTTC Seq.ID No.30CACTATGAAGAGAAGCCCCT Seq.ID No.31 AAACACAACTGCTGCAGCGT Seq.ID No.32GAAGCCAGCCGATATGGAT Seq.ID No.33 TAGAGCTAGAAGCCACTACT Seq.ID No.34TCCTGTGCAAGTACCGACCA Seq.ID No.35 CAGTAGTGGCTTCTAGCTCT Seq.ID No.36TCCTGGGCACTATGAAGAG Seq.ID No.37 GGTAGCAATATTGTAGAATCC Seq.ID No.38GTTTGTAGCACACTCCTGAT Seq.ID No.39 TATGGCTGCAGTCTGCGGT Seq.ID No.40ACTAGAGTGGTCATGGGAAC Seq.ID No.41 GATTCCAGTTTGCAAGGTAC Seq.ID No.42TACTGCTACTGCTGGGGAAT Seq.ID No.43

[0055] Amplified DNA fragments were subcloned into a TA vector andnucleotide sequences of the relp gene fragments were obtained bysequencing with vector-derived and relp specific primers. Comparison ofgenomic RELP DNA with the RELP cDNA sequence revealed that thetranscribed regions are divided into seven exons separated by sixintrons and that all exon-intron junctions followed the GT-AG rule. Thelengths of exons 1,2,3,4,5,6,7 are of 172,174,161,98,137,106 and 669 bprespectively (FIG. 4). It was determined that due to differentialsplicing exon 2 is not represented in all transcripts. The initiation ofthe first exon was deduced from the genomic sequence using the AG ruleand the splice donor acceptor site consensus sequence location. Exons 1to 3 encode the 5′ untranslated region of 440 nt (or 266 nt in thesplice variants where the exon 2 is missing) and exon 7 the 3′untranslated region of 601 nt.

[0056] The promoter sequence of the relp gene was analyzed with thepromoter analyzing program Genomatix (http:genomatix.gsf.de/mat_fam). AnAp-1 binding site and a cAMP responsive element are located at 15respectively 44 base pairs upstream from transcriptional initiationsite.

Example 7 Fluorescence in situ Hybridization (FISH)

[0057] To determine the chromosomal localization of the relp gene,fluorescent in situ hybridization (FISH) was performed. A human genomicPAC clone containing the RELP gene was used as a probe to localize RELPin human chromosomes. The PAC plasmid was labeled with biotin-16-dUTPusing nick translation. Slides with human interphase and metaphasenuclei were pretreated with 0.01 N HCl for 10 min at 37° C. and 0.01 NHCl containing pepsin (20 mg/ml) for 5 min at 37° C. After dehydrationin graded ethanol, the slides were denaturated in 70% formamide/2×SSC at64° C. Hybridization was carried out at 37° C. overnight. Afterhybridization, the slides were washed in 2×SSC for 1×5 min at 45° C.,0.1×SSC for 2×5 min at 45° C. and in 4×SSC/0.2% Tween 1×5 min at roomtemperature, blocked in 5% BSA/4×SSC for 30 min at 37° C. and in4×SSC/0.2% Tween for 5 min at 45° C. Hybridized probes were detectedwith avidin-conjugated FITC and the signals were amplified withbiotinylated-anti-avidin antibodies. After washing at 45° C. in4×SSC/0.2% Tween for 3×5 min the slides were counterstained with DAPIand mounted in an antifade solution.

[0058] Hybridization showed exclusive signals on chromosome 1 bandp12-13.1.

Example 8 Dot Blot and Northern Blot Analysis

[0059] Dot blot and Northern blot analyses were performed using MultipleTissue Expression (MTE) Array and Multiple Tissue Northern (MTN) blot IIand III (Clontech,). ³²P-labeled full length RELP cDNA was used as aprobe. Labeling was done with the Multiprime DNA labeling system kit(Amersham Pharmacia Biotech). For autoradiography filters were exposedto Kodak Biomax MS film for 1-3 days. Dot blot analysis revealed RELPmRNA in tissues of the gastrointestinal tract, in the prostate, and intestis. Northern blot analysis demonstrated high expression of a 1.5-kbtranscript in the duodenum, stomach, testis, and prostate. Significantexpression was also seen in the jejunum, ileum, ilocecum, appendix,descending colon and pancreas. No RELP expression was seen in thyroid,spinal cord, adrenal gland, bone marrow, spleen, thymus, ovary or bloodleukocytes.

[0060] The above is the description of the normal tissue distribution ofRELP.

[0061] In the cancers identified in the body of the specification above,RELP is expressed ectopically, meaning that it is expressed in cellswhich should not express it at all, where its expression is irrelevant,and is due to the regression of the level of differentiation. Thus, thepresence of RELP beyond normal levels is seen at the level of the wholeorganism: the body produces too much RELP (measured in plasma), whichindicates that there is a cancer in one of the organs known to developRELP-positive tumors.

Example 9 Reverse Transcription Polymerase Chain Reaction (RT-PCR)

[0062] Reverse transcription and PCR amplification of RELP mRNA wasperformed by continous RT-PCR using the Robust RT-PCR kit (Finnzymes,Espoo, Finland). One hundred ng of poly(A) RNA was reverse-transcribedinto cDNA for one RT-PCR reaction. The primers used were as follows:sense: CAGCTGTGCTCCTGGATGGT, Seq.ID No.12 CTCCTATTGCTGAGCTGCCT Seq.IDNo.14 antisense: TGGTCGGTACTTGCACAGGA, Seq.ID No.44 ATTCGTTGCTGCTCCAAGTTSeq.ID No.45

[0063] Reverse transcription reaction was performed at 48° C. for 30min. Before PCR amplification, the samples were initially denatured at95° C. for 4 min. Cycling parameters were as follows (30×): denaturationat 95° C. for 30 s, annealing at 60° C. for 1 min, elongation at 72° C.for 1 min and final extension at 72° C. for 5 min.

[0064] Amplified products were analyzed by agarose electrophoresis andsubcloned according to manufacturer's instructions into a vector of theTA cloning system (Invitrogen, San Diego). Nucleotide sequencing of thecloned PCR products were performed by the Thermo Sequenace Kit(Amersham, Buckingshire, UK) and an ALF express sequenator (Pharmacia,Uppsala, Sweden). The procedure verified the transcription of RELP induodenum, colon, stomach, and pancreas, and excluded the possibilitythat the Northern blot and Dot blot experiments should have detected RNArepresenting other reg proteins that are homologus to RELP.

Example 10 In vitro Translation

[0065] A cDNA fragment containing the full length sequence of RELP cDNAwas subcloned into the eukaryotic expression vector pcDNA 3 (Invitrogen,San Diego) under the T7 RNA polymerase promoter. The RELP protein wasexpressed using Rabbit Reticulocyte Lysate with Canine PancreaticMicrosomal Membranes (Promega, Madison, Wis.) in the presence of³⁵S-methionine (Amersham International's Redivue L-35Smethionine,Amersham Pharmacia Biotech). Proteins obtained by in vitro translationwere analyzed by SDS-PAGE (12%) gel electrophoresis and visualized byautoradiography. The translation resulted in a protein product with anapparent molecular weight of 18 kd as analysed by PAGE. This is inconcordance with the calculated molecular weight of RELP (18.2 kd). Whenthe microsomal membrane fraction was added, the size of the proteinproduct was reduced to 17 kd, which is in concordance with the predictedstructure of RELP, including an N-terminal cleavable 23 amino acidsignal peptide.

Example 11 Enzyme Immunoassay (Prophetic)

[0066] Immunoassays are prepared for the RELP antigen. This isachievable since detection of 10 fmol/L is possible in competitiveassays. Sensitivity of noncompetitive assay is determined by lower limitof detection of the label used: 1 to 2,000,000 Zeptomoles (10⁻²¹ moles).Tietz Fundamentals of Clinical Chemistry” 4th Edition, p143

[0067] To develop an Enzyme Immunoassay (EIA) procedure, antigenstandards comprising a digest of colon tumor specimens (shown to containthe antigen by immunoperoxidase staining) are used. Human primary coloncancer specimens are pooled and homogenized in 10 volumes of 10 mM Trisbuffer, pH 7.4, containing 0.2% (w/v) sodium deoxycholate at 4C. Thehomogenate is quickly brought to 37 C and the following reagents (finalconcentration) are added while stirring: 1 mM cysteine (Sigma), 1 mMEDTA (Sigma), and papain (0.8 unit/ml) (Boehringer-Mannheim,Indianapolis, Ind.). After 5 minutes, digestion is stopped by theaddition of 5 mM iodoacetamide (Sigma). The homogenate is centrifuged at100,000×g for 1 hour at 4C, then extensively dialyzed against 10 mMTris/0.9% NaCl solution buffer, pH 7.4, containing phenylmethysulfonylfluoride and aminocaproic acid, each at 10 mM. The homogenate is frozenin small aliquots at a concentration of 0.5 mg of protein/ml.

[0068] The dose response curve that will be generated for theimmunoassay procedure measuring RELP demonstrates linearity betweenantigen input of 100 ng to 100 μg/ml. For serum analysis, the range is 1ng to 1000 ng/ml, since these samples are diluted 10-fold prior toassay.

[0069] Solid-phase preparations of the antibodies described in Example 2are prepared using CNBr-activated Sepharose (Pharmacia). Microtiterplates (Nunc I Immunoplates; Grand Island Biological Co., Grand Island,N.Y.) are coated with the antibodies (200 μl/well) in 50 mMcarbonate-bicarbonate buffer, pH 9.6, for 18 hours at 4C. After removalof the antibody solution, residual protein binding sites on the plasticare blocked by the addition of 200 μl of assay buffer [PBS containing 1%(v/v) rabbit serum and 1% (w/v) bovine albumin]. After 1 hour ofincubation at room temperature, the coated plates are used immediatelyfor the assay procedure.

[0070] To perform the assay, 200 μl samples, diluted in assay buffer,are applied for 1-5 hours at 37C. After 3 washes using assay buffer, 200μl of the antibody covalently conjugated to horseradish peroxidase(Sigma, Type VI) is applied to each well for 1.5 hours at 37C. Theconjugate is diluted to a concentration of 0.5 μg of immunoglobulin perml of PBS containing 10% (v/v) murine serum. Following a wash procedureas above, 200 μl of substrate per well are applied for 0.5 hours at roomtemperature. Substrate solution contains 0.4 mg of o-phenylenediamineper ml of pH 5.0 citrate buffer and 0.003% hydrogen peroxide. Thereaction is stopped by addition of 50 μl of 2N sulfuric acid, andabsorbance is monitored at 488 nM using an enzyme assay plate reader(Fisher Scientific Co., Pittsburgh, Pa.).

[0071] The percentage of bound enzyme conjugate is calculated by theformula:

(B−B₀)(B_(t)−B₀)(100)

[0072] where B=absorbance of the sample, B_(t)=maximal absorbance, andB₀=absorbance of the blank. Each assay is performed in triplicate usinga standard digest and 26-fold diluted serum samples diluted in assaybuffer. Specificity of the immunoassay is examined by substitutingvarious antibody reagents at the solid phase, including an antibody toCEA and nonimmune rabbit serum. Of the solid phase antibodies onlyantibody prepared according to Example 2 binds antigen at highdilutions.

[0073] Levels of serum RELP are detected for normal control subjects,patients with benign and malignant prostate diseases and patients withovarian, stomach, colon, and breast cancer.

[0074] Sera obtained from apparently healthy individuals exhibits a meanvalue of approximately 90 ng/ml of RELP/ml. Only 5% of the samplesexpress serum antigen at 150 ng/ml or above, and this value is chosen asthe cutoff for elevated serum levels.

[0075] Sera from patients with benign disease of the colon exhibit amean RELP value of 160 ng/ml (Table IV). The incidence of values above200 ng/ml is 5%. Patients with colon cancer (with evidence of disease)exhibit a wide range of circulating levels of RELP with a mean valueabove 160 ng/ml.

[0076] Sera obtained from patients with cancers corresponding to thosedescribed above are also evaluated. The incidence of elevated RELPvalues is 90%. Mean serum values from the group with cancer aresignificantly higher than control levels (about 250% higher).

[0077] Using a limited number of postoperative colon cancer patientswith primary localized disease, a significant decrease in serum RELPoccurs. These data indicate a relationship between serum RELP levels andtumor load. Such measurements are thus valuable for patient monitoring.

1 45 1 477 DNA Human 1 atggcttcca gaagcatgcg gctgctccta ttgctgagctgcctggccaa aacaggagtc 60 ctgggtgata tcatcatgag acccagctgt gctcctggatggttttacca caagtccaat 120 tgctatggtt acttcaggaa gctgaggaac tggtctgatgccgagctcga gtgtcagtct 180 tacggaaacg gagcccacct ggcatctatc ctgagtttaaaggaagccag caccatagca 240 gagtacataa gtggctatca gagaagccag ccgatatggattggcctgca cgacccacag 300 aagaggcagc agtggcagtg gattgatggg gccatgtatctgtacagatc ctggtctggc 360 aagtccatgg gtgggaacaa gcactgtgct gagatgagctccaataacaa ctttttaact 420 tggagcagca acgaatgcaa caagcgccaa cacttcctgtgcaagtaccg accatag 477 2 158 PRT Human 2 Met Ala Ser Arg Ser Met Arg LeuLeu Leu Leu Leu Ser Cys Leu Ala 1 5 10 15 Lys Thr Gly Val Leu Gly AspIle Ile Met Arg Pro Ser Cys Ala Pro 20 25 30 Gly Trp Phe Tyr His Lys SerAsn Cys Tyr Gly Tyr Phe Arg Lys Leu 35 40 45 Arg Asn Trp Ser Asp Ala GluLeu Glu Cys Gln Ser Tyr Gly Asn Gly 50 55 60 Ala His Leu Ala Ser Ile LeuSer Leu Lys Glu Ala Ser Thr Ile Ala 65 70 75 80 Glu Tyr Ile Ser Gly TyrGln Arg Ser Gln Pro Ile Trp Ile Gly Leu 85 90 95 His Asp Pro Gln Lys ArgGln Gln Trp Gln Trp Ile Asp Gly Ala Met 100 105 110 Tyr Leu Tyr Arg SerTrp Ser Gly Lys Ser Met Gly Gly Asn Lys His 115 120 125 Cys Ala Glu MetSer Ser Asn Asn Asn Phe Leu Thr Trp Ser Ser Asn 130 135 140 Glu Cys AsnLys Arg Gln His Phe Leu Cys Lys Tyr Arg Pro 145 150 155 3 78 DNA Human 3atggcttcca gaagcatgcg gctgctccta ttgctgagct gcctggccaa aacaggagtc 60ctgggtgata tcatcatg 78 4 26 PRT Human 4 Met Ala Ser Arg Ser Met Arg LeuLeu Leu Leu Leu Ser Cys Leu Ala 1 5 10 15 Lys Thr Gly Val Leu Gly AspIle Ile Met 20 25 5 17 PRT Human 5 Cys Ala Glu Met Ser Ser Asn Asn AsnPhe Leu Thr Trp Ser Ser Asn 1 5 10 15 Glu 6 25 PRT Human 6 Cys Tyr GlyTyr Phe Arg Lys Leu Arg Asn Trp Ser Asp Ala Glu Leu 1 5 10 15 Glu CysGln Ser Tyr Gly Asn Gly Ala 20 25 7 23 PRT Human 7 Trp Ile Asp Gly AlaMet Tyr Leu Tyr Arg Ser Trp Ser Gly Lys Ser 1 5 10 15 Met Gly Gly AsnLys His Cys 20 8 17 PRT Human 8 Cys Ala Glu Met Ser Ser Asn Asn Asn PheLeu Thr Trp Ser Ser Asn 1 5 10 15 Glu 9 29 PRT Human 9 Cys Ala Glu MetSer Ser Asn Asn Asn Phe Leu Thr Trp Ser Ser Asn 1 5 10 15 Glu Cys AsnLys Arg Gln His Phe Leu Cys Lys Tyr Arg 20 25 10 27 PRT Human 10 Cys GluTyr Ile Ser Gly Tyr Gln Arg Ser Gln Pro Ile Trp Ile Gly 1 5 10 15 LeuHis Asp Pro Gln Lys Arg Gln Gln Trp Gln 20 25 11 23 PRT Human 11 Cys GlnSer Tyr Gly Asn Gly Ala His Leu Ala Ser Ile Leu Ser Leu 1 5 10 15 LysGlu Ala Ser Thr Ile Ala 20 12 20 DNA primer 12 cagctgtgct cctggatggt 2013 20 DNA primer 13 tggtcggtac ttgcacagga 20 14 20 DNA primer 14ctcctattgc tgagctgcct 20 15 20 DNA primer 15 attcgttgct gctccaagtt 20 1619 DNA primer 16 ttccagaagc atgcggctg 19 17 19 DNA primer 17 acaggaagtgttggcgctt 19 18 19 DNA primer 18 atggcttcca gaagcatgc 19 19 20 DNAprimer 19 ctatggtcgg tacttgcaca 20 20 20 DNA primer 20 cttgctctatggtcggtact 20 21 21 DNA primer 21 actgggacca ctggagacac t 21 22 19 DNAprimer 22 gagacactga agaaggcag 19 23 20 DNA primer 23 agacccagctgtttcatagg 20 24 20 DNA primer 24 aatggagaga gggcagaagg 20 25 23 DNAprimer 25 tgatatcatc atgagaccca gct 23 26 21 DNA primer 26 agacagtcatccatttgccc a 21 27 21 DNA primer 27 tgggcaaatg gatgactgtc t 21 28 21 DNAprimer 28 ctctagaatc caacaaaact c 21 29 21 DNA primer 29 tgccagaccaggatctgtac a 21 30 19 DNA primer 30 atccatatcg gctggcttc 19 31 20 DNAprimer 31 cactatgaag agaagcccct 20 32 20 DNA primer 32 aaacacaactgctgcagcgt 20 33 19 DNA primer 33 gaagccagcc gatatggat 19 34 20 DNAprimer 34 tagagctaga agccactact 20 35 20 DNA primer 35 tcctgtgcaagtaccgacca 20 36 21 DNA primer 36 cagtagtggc ttctagctct t 21 37 18 DNAprimer 37 cctgggcact atgaagag 18 38 21 DNA primer 38 ggtagcaatattgtagaatc c 21 39 20 DNA primer 39 gtttgtagca cactcctgat 20 40 19 DNAprimer 40 tatggctgca gtctgcggt 19 41 20 DNA primer 41 actagagtggtcatgggaac 20 42 20 DNA primer 42 gattccagtt tgcaaggtac 20 43 20 DNAprimer 43 tactgctact gctggggaat 20 44 20 DNA primer 44 tggtcggtacttgcacagga 20 45 20 DNA primer 45 attcgttgct gctccaagtt 20

I claim:
 1. A method of detecting the presence of a tumor comprisingdetecting the presence or quantity of: a) a nucleic acid moleculeencoding a protein having at least a 70% identity to a polypeptidecomprising amino acids of SEQ ID NO:2; b) a nucleic acid molecule whichis complementary to the nucleic acid sequence of (a); c) a nucleic acidmolecule comprising at least 15 sequential bases of the nucleic acid d)a nucleic acid molecule that hybridizes under stringent conditions tothe nucleic acid sequence molecule of (a) and correlating such quantityor presence with the presence of a tumor.
 2. A method of detecting thepresence of a tumor comprising detecting the concentration of RELP in abiological sample.
 3. The method of claim 11 further comprising the stepof correlating the presence or quantity of such molecule with thepresence or absence of said tumor.
 4. A kit for determining the presenceof RELP comprising: a) antibody immunologically reactive with RELPprotein, and b) reagents for detecting the presence of the antibody. 5.The kit of claim 4 further comprising instructions for use inidentifying the presence of cancer, characterizing the cancer, ormonitoring the course of treatment of cancer.
 6. A kit for for thepurpose of identifying the presence of cancer, characterizing thecancer, or monitoring the course of treatment of cancer, siad kitcomprising: a) a reagent nucleic acid molecule that is complementary aportion of the nucleic acid sequences that encode RELP wherein saidreagent nucleic acid molecule comprises at least 15 sequential bases ofthe nucleic acid sequence that encodes RELP; and b) a containercontaining said reagent nucleic acid molecule.
 7. The kit of claim 6wherein said said reagent nucleic acid molecule is selected from thegroup consisting of: Seq. ID No. 12-43.